PHAs are polyester-type organic polymer molecules produced by a variety of microorganisms. Actually, PHAs are a biodegradable thermoplastic polymer and also producible from renewable resources. Hence, some attempts have been made to industrially produce a PHA as an environmentally friendly material or biocompatible material for various industrial use.
PHAs are formed from monomer units generally called 3-hydroxyalkanoic acids which are specifically exemplified by 3-hydroxybutyric acid, 3-hydroxyvaleric acid, 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid, and 3-hydroxyalkanoic acids with a longer alkyl chain. The polymer molecules are formed by homopolymerization or copolymerization of the said 3-hydroxyalkanoic acids.
Examples of such a PHA include poly-3-hydroxybutyric acid (hereinafter abbreviated as P(3HB)) which is a homopolymer of 3-hydroxybutyric acid (hereinafter abbreviated as 3HB); a copolymer of 3-hydroxybutyric acid (3HB) and 3-hydroxyvaleric acid (hereinafter abbreviated as 3HV) (hereinafter, the copolymer is abbreviated as (3HB-co-3HV)); and a copolymer of 3HB and 3-hydroxyhexanoic acid (hereinafter abbreviated as 3HH) (hereinafter, the copolymer is abbreviated as (3 HB-co-3HH)).
Polyesters have different characteristics depending on the molecular weight. Polyesters with as high a molecular weight as possible are preferred in the case of fiber processing. Different microorganisms produce polyhydroxyalkanoates with different molecular weights ranging from 50,000 to 1,000,000 in general. Accordingly, production of a PHA with a higher molecular weight has been studied.
Non Patent Literatures 1, 2, and 3 show a production method for P(3HB) with a weight average molecular weight of higher than 10,000,000 by controlling the pH and glucose concentration in culturing of Escherichia coli introduced Ralstonia eutropha-derived genes related to PHA synthesis. These documents show that a high-molecular-weight P(3HB) has better physical properties (e.g., tensile strength and restretchability) which are important for fiber processing or others.
Non Patent Literature 4 shows that change of the PHA synthase concentration in in-vitro P(3HB) production system enables production of P(3HB) with a weight average molecular weight of 3,000,000 to 12,000,000.
Patent Literature 1 shows that in production of P(3HB) using Escherichia coli harboring an expression vector that contains a PHA synthase gene controlled by an inducible promoter, the amount of the enzyme expressed by the inducer enables control of the weight average molecular weight between 780,000 and 4,000,000.
Patent Literature 2 shows that expression of a PHA synthase gene integrated at different sites on a bacterial chromosome leads to production of PBAs with different molecular weights. In the case where an Aeromonas caviae-derived PHA synthase gene and genes producing a substrate monomer were integrated into R. eutropha chromosome, polyesters including 3-hydroxyhexanoate and 3-hydroxyoctanoate which has a molecular weight of 400,000 to 10 million was accumulated. Patent Literature 2, however, does not mention the productivity of this case.
There are also many study reports on production of P(3HB-co-3HH).
Patent Literature 3 shows production of P(3HB-co-3RH) with a weight average molecular weight of 5,100,000 at level of 109.2 g/L-biomass and 68.6% of polyester content after 64-hr cultivation of a transformant which is introduced a PHA synthase mutant gene derived from A. caviae and a 3-ketoacyl-ACP reductase gene (fabG) derived from Escherichia coli into R. eutropha, by using palm oil as an inexpensive carbon source.
The previous technologies have showed the production of high-molecular-weight P(3HB) and P(3HB-co-3HH) with a weight average molecular weight of at least 4,000,000 as described above, but employ expensive materials and have low productivity, which have been problems in achieving inexpensive industrial production of a high-molecular-weight polyester at a high yield.